CN104817565B - Method for efficient extraction of tripterygium wilfordii alkaloids by ionic liquid - Google Patents
Method for efficient extraction of tripterygium wilfordii alkaloids by ionic liquid Download PDFInfo
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- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/12—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains three hetero rings
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Abstract
The invention relates to a method for efficient extraction of tripterygium wilfordii alkaloids by ionic liquid. The method comprises the steps of: 1) ultrasonic enhanced extraction: preparing an extraction solution, i.e. a mixed solution of ethanol, water and an imidazole ionic liquid in a volume ratio of 1:0.1-2.0:0.5-2.0, controlling the ultrasonic treatment power density at 10-200W/cm<2> and the frequency at 20-100kHz; 2) organic solvent extraction: using an organic solvent to extract the tripterygium wilfordii alkaloid extraction solution; 3) recrystallization: removing an organic phase from the extraction phase, then conducting alkalization, filtration and recrystallization to obtain the tripterygium wilfordii total alksloid crude product; 4) HSCCC separation and purification: selecting an HSCCC solvent system, using an ionic liquid II as the mobile phase additive, and conducting HSCCC separation and purification to obtain a tripterygium wilfordii alkaloid solution; and 5) vacuum freeze drying. The method greatly reduces the use of the organic solvent, significantly reduces the pollution and damage in the extraction process, and greatly improves the extraction effect and extraction efficiency. Also, the ionic liquid can greatly shorten the separation time of HSCCC, and the purity of the obtained four tripterygium wilfordii alkaloids is more than 95%.
Description
Technical field
The present invention relates to technology of pharmaceutical engineering field, more particularly to it is a kind of biological using ionic liquid high efficiency extraction Radix Tripterygii Wilfordii
The method of alkali.
Background technology
Radix Tripterygii Wilfordii (Tripterygiumwilfordii Hook.f.) is Celastraceae tripterygium plant, with root, leaf, flower
And fruit is used as medicine, and is widely used to treat various autoimmune diseasees, such as nephritis, lupus erythematosus, contact dermatitis and class
The autoimmune diseasees such as rheumatic arthritis.Thunder godvine alkaloid is that the class produced in Radix Tripterygii Wilfordii plant has higher physiology
The native chemical thing of activity, because it has special chemical constitution and mechanism of action, particularly clinically to autoimmunity
Disease (such as lupus erythematosus, rheumatoid arthritiss, ankylosing spondylitises and anaphylactoid purpura) has unique curative effect, pharmacological research
Prove that it has the effects such as antiinflammatory, immunosuppressant, antitumor, suppression male fertility, anti-AIDS, parasite killing.In recent years, people couple
The chemical composition of thunder godvine alkaloid and pharmacological action have carried out substantial amounts of research, it has been found that the clear and definite Thunder God of 30 various structures
Rattan alkaloid, such as wilfortrine (wilfortrine), wilforine (wilforine), wilfordine
And the sesquiterpene Macrolide alkaloid such as Wilforgine (wilforgine) (wilfordine).Some of which has obvious
Immunosuppressant, antitumor action and less toxicity, fully show the good application prospect of thunder godvine alkaloid.Radix Tripterygii Wilfordii
The structural formula of spring alkali, wilforine, wilfordine and Wilforgine is as follows：
Note：Ac is representedThat is acetyl group
At present, the method for thunder godvine alkaloid being extracted from Radix et Rhizoma Tripterygii, stem, leaf is mainly entered using volatile organic solvent
Row is extracted, and using liquid/liquid extraction binding silica gel column chromatography and liquid/liquid extraction high-speed countercurrent chromatography (HSCCC) is combined.Such as patent
Number for ZL200610053146.8 (Authorization Notice No. be CN 100422188C) Chinese invention patent《Counter current chromatography is from thunder
The method for preparing thunder godvine alkaloid monomer is separated in Tripterygium wilfordii》, by the radical center of Radix Tripterygii Wilfordii, root bark or stem xeraphium in the invention
It is broken, it is percolated with ethanol solution, it is percolated the ethanol solution extracting solution that obtains and is evaporated to dry ethanol extract, ethanol extract Jing chlorine
Be evaporated to dry chloroform extractum after imitative extraction 3～6 times, chloroform extractum is Jing filtering, washing, after drying thick total alkaloidss,
With after recrystallizing methanol total alkaloidss, finally separated using adverse current chromatogram obtain single wilfortrine, wilfordine,
Wilforgine and wilforine.Each thunder godvine alkaloid purity that the method is obtained can reach more than 95%, but carry
Substantial amounts of organic solvent is taken and separated used in preparation process, safety and the pollution problem of organic solvent is there is.
The special performance of ionic liquid is widely paid close attention in chemical field, at present in the extraction separation of natural product
In succeed application, the such as middle promulgated by the State Council of Patent No. ZL200810114416.0 (Authorization Notice No. is CN 101597296B)
Bright patent《A kind of new method of efficiently extracting and producing artemisinin by ionic liquid》Using glyoxaline ion liquid halide salts and water
Mixture it is ultrasound-enhanced extract Herba Artemisiae Annuae raw material in arteannuin.At present, with regard to using ionic liquid from Radix et Rhizoma Tripterygii, stem, leaf
The middle method for extracting thunder godvine alkaloid has not been reported, therefore, study ionic liquid answering in thunder godvine alkaloid extraction
With the potential safety hazards to solving to be caused using organic reagent in a large number in prepared by current thunder godvine alkaloid extraction separation have heavy
Want meaning.
The content of the invention
The technical problem to be solved is to provide one kind for prior art safely, effectively to utilize ionic liquid
The method that body extracts thunder godvine alkaloid.
The present invention solve the technical scheme that adopted of above-mentioned technical problem for：One kind utilizes ionic liquid high efficiency extraction Thunder God
The method of rattan alkaloid, it is characterised in that comprise the following steps：
1) ultrasound-enhanced extraction：The drying and crushing such as Radix et Rhizoma Tripterygii, stem, leaf, cross 40～80 mesh sieves, by solid-to-liquid ratio 1:5～
It is 1 that 100g/mL adds volume ratio:0.1～2.0:0.5～2.0 ethanol, water and the mixed liquor of glyoxaline ion liquid I, in ultrasound
10～200W/cm of power density2And under 20～100kHz of supersonic frequency, 20～95 DEG C of 5～90min of supersound extraction, solid-liquid separation
The extracting solution of thunder godvine alkaloid must be contained afterwards；
2) organic solvent extraction：Add organic solvents into the step 1) in the extracting solution that obtains, by extracting solution with have
The volume ratio 1～10 of machine solvent:1 extraction 1～5 time, separates organic faciess and obtains thunder godvine alkaloid extraction phase, and extraction extraction raffinate circulation makes
With the extraction extraction raffinate is above-mentioned glyoxaline ion liquid I；
3) recrystallization：By the step 2) in the acidified process of the extraction phase Jing that obtains to remove organic faciess, and water is mutually used
Filter after sodium hydroxide solution alkalization, the precipitate being filtrated to get is re-dissolved in carry out recrystallization in methanol, obtain tripterygium total
Alkaloid crude product；
4) HSCCC is isolated and purified：HSCCC solvent systems are selected, by volume (mL)/weight (g) ratio 100～600:1 will
HSCCC solvent systems and above-mentioned tripterygium total alkaloid crude product are added in separatory funnel, concussion, stratification, after balance, will
Upper and lower phase separates, and with the upper as fixing phase of solvent system, the lower phase of solvent system is mobile phase, rotating speed is adjusted, with a constant current
Speed pumps into mobile phase, according to the spectrogram Fraction collection object of detector, each thunder godvine alkaloid solution is obtained respectively；
5) vacuum lyophilization：By above-mentioned each thunder godvine alkaloid solution, the vacuum freezing under proper temperature, drying pressure
It is dried, reclaims organic extractant, respectively obtains wilfortrine, wilforine, wilfordine that purity is all higher than 95%
And Wilforgine.
Preferably, the glyoxaline ion liquid I is 1- ethyl-3-methylimidazole villaumites, 1- ethyl-3-methylimidazoles
In bromide, 1- butyl -3- Methylimidazole. bromides, 1- octyl group -3- methylimidazole villaumites, 1- pi-allyl -3- methylimidazole villaumites
It is at least one.
Preferably, the step 1) in ethanol, water and the mixed liquor of glyoxaline ion liquid I volume ratio be 3:2:5.
Preferably, the organic solvent is in acetone, dichloromethane, ethyl acetate, chloroform and carbon tetrachloride
It is at least one.
Preferably, it is 6 that the HSCCC solvent systems are volume ratio:4:5:8:0.05 n-hexane-ethyl acetate-ethanol-
The HSCCC solvent systems of the preparation of water-ionic liquid II, volume ratio are 2:4:2:5:0.05 methyl tertiary butyl ether(MTBE)-n-butyl alcohol-second
The HSCCC solvent systems or volume ratio that nitrile-water-ionic liquid II is prepared is 5:4:3:0.05 chloroform-ethanol-water-ionic liquid
Any one in the HSCCC solvent systems that body II is prepared.
Preferably, the glyoxaline ion liquid II is 1- ethyl-3-methylimidazole tetrafluoroborates, 1- ethyl -3-
In Methylimidazole. hexafluorophosphate, 1- butyl -3- methyl imidazolium tetrafluoroborates, 1- butyl -3- Methylimidazole. hexafluorophosphates
At least one.
Compared with prior art, it is an advantage of the current invention that：The present invention is using ethanol, the water for mixing by a certain percentage
With the mixed liquor of glyoxaline ion liquid I as extracting solution, the Radix Tripterygii Wilfordii in Radix et Rhizoma Tripterygii, stem, leaf is extracted with reference to ultrasonic wave added
Alkaloid, the extracting solution greatlys save the usage amount of organic solvent, green, ring compared with existing pure organic solvent extraction
Protect, significantly reduce pollution and harm that traditional volatile organic solvent extracting method brings to environment and experimenter, together
When ionic liquid addition, substantially improve extraction effect, improve extraction efficiency.Further, with ionic liquid in the present invention
As HSCCC Mobile Phase Additives, four kinds of thunder godvine alkaloids (wilfortrine, wilforine, wilfordine and Thunder Gods
Rattan alkali second) appearance time reduce to 120min from existing 250min, keeping, various thunder godvine alkaloid separating degrees are basic
On the basis of constant (separating degree is all higher than 1.5), the disengaging time of HSCCC is substantially reduced, reached and be kept completely separate, obtained
The purity of four kinds of thunder godvine alkaloids is all higher than 95%, substantially increases production efficiency.
Description of the drawings
Fig. 1 is the ultraviolet chromatogram of the detached four kinds of thunder godvine alkaloids of HSCCC methods in the embodiment of the present invention 1, wherein,
(1) it is wilfortrine, (2) are wilfordine, (3) are Wilforgine, (4) wilforine.
Specific embodiment
The present invention is described in further detail below in conjunction with accompanying drawing embodiment.
Embodiment 1：
1) ultrasound-enhanced extraction：Radix et Rhizoma Tripterygii 1.0kg drying and crushing is taken, 80 mesh sieves are crossed, 100 grams of powder is then taken and is put into
In 2500 milliliters of conical flasks, by solid-to-liquid ratio 1:5 (W/V) add ethanol/water/1- ethyl-3-methylimidazoles villaumite (7:3:7, V/V/
V mixture), in ultrasonic power density 100W/cm2, supersonic frequency 100kHz, under the conditions of 65 DEG C of temperature, supersound extraction
30min, Jing solid-liquid separation obtains the extracting solution containing thunder godvine alkaloid.
2) organic solvent extraction：Chloroform is added to into above-mentioned steps 1) in the extracting solution that obtains, extracting solution and acetone
Volume ratio be 5:1 (V/V), extracts 2 times, separates organic faciess and obtains thunder godvine alkaloid extraction phase, and extraction extraction raffinate is recycled, should
Extraction extraction raffinate is 1- ethyl-3-methylimidazole villaumites.
3) recrystallization：By step 2) in the extraction phase that obtains remove organic faciess, water phase Jing after the hydrochloric acid acidifying of 1.0mol/L
The sodium hydroxide for adding 1.0mol/L adjusts pH to 9.5, stands and filtered after 15min, and precipitate is re-dissolved in methanol and is tied again
Crystalline substance, obtains tripterygium total alkaloid crude product.
4) HSCCC is isolated and purified：Weigh step 3) the tripterygium total alkaloid crude product 0.50g of Jing recrystallization that obtains, choosing
Select normal hexane-ethyl acetate-alcohol-water -1- ethyl-3-methylimidazoles tetrafluoroborate (6:4:5:8:0.05, v/v) conduct
HSCCC solvent systems, the HSCCC solvent systems of 200mL and tripterygium total alkaloid crude product are added in separatory funnel, concussion
Solution is sufficiently mixed, is stood overnight, after split-phase balance upper and lower phase is separated.With the upper as fixing phase of solvent system, under
It is mutually mobile phase, adjustment rotating speed is 700r/min, and column temperature is 25 DEG C, and with high-speed counter-current chromatograph, (Shanghai has with field biotechnology
The TBE-1000A types of limit company production) HSCCC serpentine pipes are pumped into 5.0mL/min flow velocitys, received according to the spectrogram substep of detector
Collection object, isolates and purifies and obtain respectively each thunder godvine alkaloid, and HSCCC separation chromatography figures are as shown in Figure 1.
5) vacuum lyophilization and detection：By step 4) collection liquid that obtains, -20 DEG C of pre-freezing temperature, operating pressure 25Pa,
55 DEG C of sublimation temperature, 65 DEG C of resolution temperature obtains wilfortrine, wilforine, wilfordine that purity is all higher than 95%
Alkali and Wilforgine.
Detection adopts Agilent 1100series high performance liquid chromatographs, and chromatographic column is Zorbax XDB C18 posts
(250mm*4.6mm i.d., 5 μm, Agilent companies), mobile phase is acetonitrile/water=60/40 (V/V), and flow velocity is 0.7mL/
Min, UV-detector is detected that the purity of each thunder godvine alkaloid is more than 95% under 230nm wavelength.
Embodiment 2：
1) ultrasound-enhanced extraction：Thunder God rattan 1.0kg drying and crushing is taken, 60 mesh sieves are crossed, 100 grams of powder is then taken and is put into
In 2500 milliliters of conical flasks, by solid-to-liquid ratio 1:10 (W/V) add ethanol/water/1- ethyl-3-methylimidazoles bromide (3:2:5, V/
V/V mixture), in ultrasonic power density 50W/cm2, supersonic frequency 50kHz, under the conditions of temperature 45 C, supersound extraction
20min, Jing solid-liquid separation obtains the extracting solution containing thunder godvine alkaloid.
2) organic solvent extraction：Chloroform is added to into above-mentioned steps 1) in the extracting solution that obtains, extracting solution and trichlorine
The volume ratio of methane is 6:1 (V/V), extracts 3 times, separates organic faciess and obtains thunder godvine alkaloid extraction phase, and extraction extraction raffinate circulation makes
With the extraction extraction raffinate is 1- ethyl-3-methylimidazole bromides.
3) recrystallization：By step 2) in the extraction phase that obtains remove organic faciess, water phase Jing after the hydrochloric acid acidifying of 1.0mol/L
The sodium hydroxide for adding 1.0mol/L adjusts pH to 10.0, stands and filtered after 15min, and precipitate is re-dissolved in methanol and is tied again
Crystalline substance, obtains tripterygium total alkaloid crude product.
4) HSCCC is isolated and purified：Weigh step 3) the tripterygium total alkaloid crude product 0.50g of Jing recrystallization that obtains, choosing
Select methyl tertiary butyl ether(MTBE)-n-butyl alcohol-acetonitrile-water -1- ethyl-3-methylimidazoles hexafluorophosphate (2:4:2:5:0.05, v/v) make
For HSCCC solvent systems, the HSCCC solvent systems of 150mL and tripterygium total alkaloid crude product are added in separatory funnel, shake
Swinging is sufficiently mixed solution, stands overnight, and after split-phase balance upper and lower phase is separated.With the upper as fixing phase of solvent system,
The lower phase of solvent system is mobile phase, and adjustment rotating speed is 700r/min, and column temperature is 25 DEG C, and with high-speed counter-current chromatograph, (Shanghai is same
The TBE-1000A types of field Bioisystech Co., Ltd production) HSCCC serpentine pipes are pumped into 5.0mL/min flow velocitys, according to detector
Spectrogram Fraction collection object, isolate and purify and obtain respectively each thunder godvine alkaloid.
5) vacuum lyophilization and detection：By step 4) collection liquid that obtains, -15 DEG C of pre-freezing temperature, operating pressure 30Pa,
50 DEG C of sublimation temperature, 60 DEG C of resolution temperature obtains wilfortrine, wilforine, wilfordine that purity is all higher than 95%
Alkali and Wilforgine.
Detection adopts Agilent 1100series high performance liquid chromatographs, and chromatographic column is Zorbax XDB C18 posts
(250mm*4.6mm i.d., 5 μm, Agilent companies), mobile phase is acetonitrile/water=60/40 (V/V), and flow velocity is 0.7mL/
Min, UV-detector is detected that the purity of each thunder godvine alkaloid is more than 95% under 230nm wavelength.
Embodiment 3：
1) ultrasound-enhanced extraction：Tripterygium leaf 1.0kg drying and crushing is taken, 40 mesh sieves are crossed, 100 grams of powder is then taken and is put into
In 2500 milliliters of conical flasks, by solid-to-liquid ratio 1：20 (W/V) add ethanol/water/1- butyl -3- Methylimidazole .s bromide (5:2:5, V/
V/V mixture), in ultrasonic power density 200W/cm2, supersonic frequency 20kHz, under the conditions of 75 DEG C of temperature, supersound extraction
60min, Jing solid-liquid separation obtains the extracting solution containing thunder godvine alkaloid.
2) organic solvent extraction：Chloroform is added to into above-mentioned steps 1) in the extracting solution that obtains, extracting solution and dichloro
The volume ratio of methane is 1:1 (V/V), extracts 5 times, separates organic faciess and obtains thunder godvine alkaloid extraction phase, and extraction extraction raffinate circulation makes
With the extraction extraction raffinate is 1- butyl -3- Methylimidazole. bromides.
3) recrystallization：By step 2) in the extraction phase that obtains remove organic faciess, water phase Jing after the hydrochloric acid acidifying of 0.5mol/L
The sodium hydroxide for adding 0.5mol/L adjusts pH to 10.0, stands and filtered after 15min, and precipitate is re-dissolved in methanol and is tied again
Crystalline substance, obtains tripterygium total alkaloid crude product.
4) HSCCC is isolated and purified：Weigh step 3) the tripterygium total alkaloid crude product 0.50g of Jing recrystallization that obtains, choosing
Select chloroform-ethanol-water -1- butyl -3- methyl imidazolium tetrafluoroborates and prepare (5:4:3:0.05, v/v) as HSCCC solvents system
System, the HSCCC solvent systems of 250mL and tripterygium total alkaloid crude product are added in separatory funnel, and concussion makes solution abundant
Mixing, stands overnight, and after split-phase balance upper and lower phase is separated.With the upper as fixing phase of solvent system, under solvent system
It is mutually mobile phase, adjustment rotating speed is 700r/min, and column temperature is 25 DEG C, and with high-speed counter-current chromatograph, (Shanghai has with field biotechnology
The TBE-1000A types of limit company production) HSCCC serpentine pipes are pumped into 5.0mL/min flow velocitys, received according to the spectrogram substep of detector
Collection object, isolates and purifies and obtain respectively each thunder godvine alkaloid.
5) vacuum lyophilization and detection：By step 4) collection liquid that obtains, -25 DEG C of pre-freezing temperature, operating pressure 25Pa,
50 DEG C of sublimation temperature, 60 DEG C of resolution temperature obtains wilfortrine, wilforine, wilfordine that purity is all higher than 95%
Alkali and Wilforgine.
Detection adopts Agilent 1100series high performance liquid chromatographs, and chromatographic column is Zorbax XDB C18 posts
(250mm*4.6mm i.d., 5 μm, Agilent companies), mobile phase is acetonitrile/water=60/40 (V/V), and flow velocity is 0.7mL/
Min, UV-detector is detected that the purity of each thunder godvine alkaloid is more than 95% under 230nm wavelength.
Embodiment 4：
1) ultrasound-enhanced extraction：Radix et Rhizoma Tripterygii 1.0kg drying and crushing is taken, 80 mesh sieves are crossed, 100 grams of powder is then taken and is put into
In 2500 milliliters of conical flasks, by solid-to-liquid ratio 1:50 (W/V) add ethanol/water/1- octyl group -3- methylimidazole villaumites (10:1:5, V/
V/V mixture), in ultrasonic power density 20W/cm2, supersonic frequency 70kHz, under the conditions of 95 DEG C of temperature, supersound extraction
90min, Jing solid-liquid separation obtains the extracting solution containing thunder godvine alkaloid.
2) organic solvent extraction：Chloroform is added to into above-mentioned steps 1) in the extracting solution that obtains, extracting solution and acetic acid
The volume ratio of ethyl ester is 10:1 (V/V), extracts 4 times, separates organic faciess and obtains thunder godvine alkaloid extraction phase, and extraction extraction raffinate circulation makes
With the extraction extraction raffinate is 1- octyl group -3- methylimidazole villaumites.
3) recrystallization：By step 2) in the extraction phase that obtains remove organic faciess, water phase Jing after the hydrochloric acid acidifying of 1.0mol/L
The sodium hydroxide for adding 1.0mol/L adjusts pH to 9.5, stands and filtered after 15min, and precipitate is re-dissolved in methanol and is tied again
Crystalline substance, obtains tripterygium total alkaloid crude product.
4) HSCCC is isolated and purified：Weigh step 3) the tripterygium total alkaloid crude product 0.50g of Jing recrystallization that obtains, choosing
Select normal hexane-ethyl acetate-alcohol-water -1- ethyl-3-methylimidazole hexafluoros borate (6:4:5:8:0.05, v/v) conduct
HSCCC solvent systems, the HSCCC solvent systems of 300mL and tripterygium total alkaloid crude product are added in separatory funnel, concussion
Solution is sufficiently mixed, is stood overnight, after split-phase balance upper and lower phase is separated.With the upper as fixing phase of solvent system, under
It is mutually mobile phase, adjustment rotating speed is 700r/min, and column temperature is 25 DEG C, and with high-speed counter-current chromatograph, (Shanghai has with field biotechnology
The TBE-1000A types of limit company production) HSCCC serpentine pipes are pumped into 5.0mL/min flow velocitys, received according to the spectrogram substep of detector
Collection object, isolates and purifies and obtain respectively each thunder godvine alkaloid.
5) vacuum lyophilization and detection：By step 4) collection liquid that obtains, -20 DEG C of pre-freezing temperature, operating pressure 25Pa,
55 DEG C of sublimation temperature, 65 DEG C of resolution temperature obtains wilfortrine, wilforine, wilfordine that purity is all higher than 95%
Alkali and Wilforgine.
Detection adopts Agilent 1100series high performance liquid chromatographs, and chromatographic column is Zorbax XDB C18 posts
(250mm*4.6mm i.d., 5 μm, Agilent companies), mobile phase is acetonitrile/water=60/40 (V/V), and flow velocity is 0.7mL/
Min, UV-detector is detected that the purity of each thunder godvine alkaloid is more than 95% under 230nm wavelength.
Embodiment 5：
1) ultrasound-enhanced extraction：Thunder God rattan 1.0kg drying and crushing is taken, 60 mesh sieves are crossed, 100 grams of powder is then taken and is put into
In 2500 milliliters of conical flasks, by solid-to-liquid ratio 1:70 (W/V) add ethanol/water/1- pi-allyl -3- methylimidazole villaumites (1:1:1,
V/V/V mixture), in ultrasonic power density 10W/cm2, supersonic frequency 200kHz, under the conditions of 25 DEG C of temperature, supersound extraction
70min, Jing solid-liquid separation obtains the extracting solution containing thunder godvine alkaloid.
2) organic solvent extraction：Chloroform is added to into above-mentioned steps 1) in the extracting solution that obtains, extracting solution and tetrachloro
The volume ratio for changing carbon is 7:1 (V/V), extracts 1 time, separates organic faciess and obtains thunder godvine alkaloid extraction phase, and extraction extraction raffinate circulation makes
With the extraction extraction raffinate is 1- pi-allyl -3- methylimidazole villaumites.
3) recrystallization：By step 2) in the extraction phase that obtains remove organic faciess, water phase Jing after the hydrochloric acid acidifying of 1.0mol/L
The sodium hydroxide for adding 1.0mol/L adjusts pH to 10.0, stands and filtered after 15min, and precipitate is re-dissolved in methanol and is tied again
Crystalline substance, obtains tripterygium total alkaloid crude product.
4) HSCCC is isolated and purified：Weigh step 3) the tripterygium total alkaloid crude product 0.50g of Jing recrystallization that obtains, choosing
Select methyl tertiary butyl ether(MTBE)-n-butyl alcohol-acetonitrile-water -1- ethyl-3-methylimidazoles tetrafluoroborate (2:4:2:5:0.05, v/v) make
For HSCCC solvent systems, the HSCCC solvent systems of 200mL and tripterygium total alkaloid crude product are added in separatory funnel, shake
Swinging is sufficiently mixed solution, stands overnight, and after split-phase balance upper and lower phase is separated.With the upper as fixing phase of solvent system,
The lower phase of solvent system is mobile phase, and adjustment rotating speed is 700r/min, and column temperature is 25 DEG C, and with high-speed counter-current chromatograph, (Shanghai is same
The TBE-1000A types of field Bioisystech Co., Ltd production) HSCCC serpentine pipes are pumped into 5.0mL/min flow velocitys, according to detector
Spectrogram Fraction collection object, isolate and purify and obtain respectively each thunder godvine alkaloid.
5) vacuum lyophilization and detection：By step 4) collection liquid that obtains, -15 DEG C of pre-freezing temperature, operating pressure 30Pa,
50 DEG C of sublimation temperature, 60 DEG C of resolution temperature obtains wilfortrine, wilforine, wilfordine that purity is all higher than 95%
Alkali and Wilforgine.
Detection adopts Agilent 1100series high performance liquid chromatographs, and chromatographic column is Zorbax XDB C18 posts
(250mm*4.6mm i.d., 5 μm, Agilent companies), mobile phase is acetonitrile/water=60/40 (V/V), and flow velocity is 0.7mL/
Min, UV-detector is detected that the purity of each thunder godvine alkaloid is more than 95% under 230nm wavelength.
Embodiment 6：
1) ultrasound-enhanced extraction：Tripterygium leaf 1.0kg drying and crushing is taken, 40 mesh sieves are crossed, 100 grams of powder is then taken and is put into
In 2500 milliliters of conical flasks, by solid-to-liquid ratio 1:100 (W/V) add ethanol/water/1- butyl -3- Methylimidazole .s bromide (5:2:5, V/
V/V mixture), in ultrasonic power density 200W/cm2, supersonic frequency 100kHz, under the conditions of 95 DEG C of temperature, supersound extraction
5min, Jing solid-liquid separation obtains the extracting solution containing thunder godvine alkaloid.
2) organic solvent extraction：Chloroform is added to into above-mentioned steps 1) in the extracting solution that obtains, extracting solution and trichlorine
The volume ratio of methane is 6:1 (V/V), extracts 5 times, separates organic faciess and obtains thunder godvine alkaloid extraction phase, and extraction extraction raffinate circulation makes
With the extraction extraction raffinate is 1- butyl -3- Methylimidazole. bromides.
3) recrystallization：By step 2) in the extraction phase that obtains remove organic faciess, water phase Jing after the hydrochloric acid acidifying of 0.5mol/L
The sodium hydroxide for adding 0.5mol/L adjusts pH to 10.0, stands and filtered after 15min, and precipitate is re-dissolved in methanol and is tied again
Crystalline substance, obtains tripterygium total alkaloid crude product.
4) HSCCC is isolated and purified：Weigh step 3) the tripterygium total alkaloid crude product 0.50g of Jing recrystallization that obtains, choosing
Select chloroform-ethanol-water -1- butyl -3- methyl imidazolium tetrafluoroborates and prepare (5:4:3:0.05, v/v) as HSCCC solvents system
System, the HSCCC solvent systems of 50mL and tripterygium total alkaloid crude product are added in separatory funnel, and concussion makes solution fully mix
Close, stand overnight, after split-phase balance upper and lower phase is separated.With the upper as fixing phase of solvent system, the lower phase of solvent system
For mobile phase, adjustment rotating speed is 700r/min, and column temperature is 25 DEG C, and with high-speed counter-current chromatograph, (Shanghai is limited with field biotechnology
The TBE-1000A types of company's production) HSCCC serpentine pipes are pumped into 5.0mL/min flow velocitys, according to the spectrogram Fraction collection of detector
Object, isolates and purifies and obtain respectively each thunder godvine alkaloid.
5) vacuum lyophilization and detection：By step 4) collection liquid that obtains, -25 DEG C of pre-freezing temperature, operating pressure 25Pa,
50 DEG C of sublimation temperature, 60 DEG C of resolution temperature obtains wilfortrine, wilforine, wilfordine that purity is all higher than 95%
Alkali and Wilforgine.
Detection adopts Agilent 1100series high performance liquid chromatographs, and chromatographic column is Zorbax XDB C18 posts
(250mm*4.6mm i.d., 5 μm, Agilent companies), mobile phase is acetonitrile/water=60/40 (V/V), and flow velocity is 0.7mL/
Min, UV-detector is detected that the purity of each thunder godvine alkaloid is more than 95% under 230nm wavelength.
Claims (3)
1. a kind of method of utilization ionic liquid extract thunder godvine alkaloid, it is characterised in that comprise the following steps：
1) ultrasound-enhanced extraction：Radix et Rhizoma Tripterygii, stem, leaf drying and crushing, cross 40～80 mesh sieves, by solid-to-liquid ratio 1:5～100g/mL adds
Enter volume ratio for 1:0.1～2.0:0.5～2.0 ethanol, water and the mixed liquor of glyoxaline ion liquid I, in ultrasonic power density
10～200W/cm2And under 20～100kHz of supersonic frequency, 20～95 DEG C of 5～90min of supersound extraction, solid-liquid separation must contain Thunder God
The extracting solution of rattan alkaloid, the glyoxaline ion liquid I is 1- ethyl-3-methylimidazole villaumites, 1- ethyl-3-methylimidazoles
In bromide, 1- butyl -3- Methylimidazole. bromides, 1- octyl group -3- methylimidazole villaumites, 1- pi-allyl -3- methylimidazole villaumites
It is at least one；
2) organic solvent extraction：Add organic solvents into the step 1) in the extracting solution that obtains, it is molten with organic by extracting solution
The volume ratio 1～10 of agent:1 extraction 1～5 time, separates organic faciess and obtains thunder godvine alkaloid extraction phase, and extraction extraction raffinate is recycled,
The extraction extraction raffinate is above-mentioned glyoxaline ion liquid I；
3) recrystallization：By the step 2) in the acidified process of the extraction phase Jing that obtains to remove organic faciess, and water mutually uses hydrogen-oxygen
Change and filtered after sodium solution alkalization, the precipitate being filtrated to get is re-dissolved in carry out recrystallization in methanol, obtain tripterygium total biological
Alkali crude product；
4) HSCCC is isolated and purified：HSCCC solvent systems are selected, by volume/weight ratio 100～600:1mL/g is by HSCCC solvents
System and above-mentioned tripterygium total alkaloid crude product are added in separatory funnel, concussion, stratification, after balance, by upper and lower phase
Separate, with the upper as fixing phase of solvent system, the lower phase of solvent system is mobile phase, adjusts rotating speed, is pumped into certain flow rate
Mobile phase, according to the spectrogram Fraction collection object of detector, obtains respectively each thunder godvine alkaloid solution, and the HSCCC is molten
It is 6 that agent system is volume ratio:4:5:8:What 0.05 n-hexane-ethyl acetate-alcohol-water-glyoxaline ion liquid II was prepared
HSCCC solvent systems, volume ratio are 2:4:2:5:0.05 methyl tertiary butyl ether(MTBE)-n-butyl alcohol-acetonitrile-water-glyoxaline ion liquid
The II HSCCC solvent systems prepared or volume ratio are 5:4:3:0.05 chloroform-ethanol-water-glyoxaline ion liquid II is prepared
HSCCC solvent systems in any one, the glyoxaline ion liquid II be 1- ethyl-3-methylimidazole Tetrafluoroboric acids
Salt, 1- ethyl-3-methylimidazole hexafluorophosphates, 1- butyl -3- methyl imidazolium tetrafluoroborates, 1- butyl -3- Methylimidazole .s
At least one in hexafluorophosphate；
5) vacuum lyophilization：By above-mentioned each thunder godvine alkaloid solution, vacuum freezing is done under proper temperature, drying pressure
It is dry, reclaim organic extractant, respectively obtain purity be all higher than 95% wilfortrine, wilforine, wilfordine and
Wilforgine.
2. as claimed in claim 1 using the method for ionic liquid extract thunder godvine alkaloid, it is characterised in that：The step
1) volume ratio of ethanol, water and the mixed liquor of glyoxaline ion liquid I is 3 in:2:5.
3. as claimed in claim 1 using the method for ionic liquid extract thunder godvine alkaloid, it is characterised in that：It is described organic
Solvent is at least one in acetone, dichloromethane, ethyl acetate, chloroform and carbon tetrachloride.
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